WO2020235668A1 - Method for screening component that affects fluctuation of adhesion proteins in stratum corneum cells - Google Patents

Method for screening component that affects fluctuation of adhesion proteins in stratum corneum cells Download PDF

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Publication number
WO2020235668A1
WO2020235668A1 PCT/JP2020/020229 JP2020020229W WO2020235668A1 WO 2020235668 A1 WO2020235668 A1 WO 2020235668A1 JP 2020020229 W JP2020020229 W JP 2020020229W WO 2020235668 A1 WO2020235668 A1 WO 2020235668A1
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screening
stratum corneum
sample
component
slide
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PCT/JP2020/020229
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French (fr)
Japanese (ja)
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多田　明弘
史佳 武重
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ポーラ化成工業株式会社
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Priority to CN202080037961.3A priority Critical patent/CN113874704A/en
Priority to SG11202112996PA priority patent/SG11202112996PA/en
Priority to JP2021520870A priority patent/JPWO2020235668A1/ja
Publication of WO2020235668A1 publication Critical patent/WO2020235668A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for screening components that affect the increase or decrease of adhesive proteins between stratum corneum cells, and a method for preparing a stratum corneum cell sample suitable for the screening method.
  • Desmosome is a typical adhesive device for keratinocytes (keratinocytes). Desmosomes are structures involved in the adhesion between epidermal cells and stratum corneum cells, and the adhesion between epidermal cells via desmosomes cooperates with the cytoskeleton consisting of keratin fibers to maintain a strong structure in epithelial tissue. It is known to be done.
  • Desmoglein 1, 2, 3, 4, desmocollin 1, 2, 3, and desmoplakin 1, 2, etc. are known as constituent proteins of this desmosome.
  • desmoglein 1 which is an adhesive protein, has a close correlation between proteolysis of desmosome components and cell detachment. Therefore, confirming the presence state of desmoglein in the stratum corneum is also an evaluation of desmosome function, and is extremely important in evaluating the state of the skin.
  • a stratum corneum cell sample is prepared by collecting stratum corneum cells using a tape stripping method and transferring to a slide glass, and an adhesive protein is stained by an immune tissue staining method, and any There is a method of observing with a microscope.
  • An object of the present invention is to provide a method for screening components that affect the increase or decrease of adhesive proteins existing between stratum corneum cells such as desmoglein, which is more accurate than the conventional method.
  • a further object of the present invention is to provide a method for preparing a stratum corneum cell sample for more accurately evaluating the abundance of an adhesive protein in the above-mentioned screening method.
  • the present invention that solves the above problems
  • a screening method for components that affect the increase or decrease of adhesive proteins between stratum corneum cells A collection process for collecting stratum corneum cells using the tape stripping method, A transfer process that transfers the stratum corneum onto a glass slide whose surface is positively charged, A cleaning process for cleaning the slide glass and A preparation step of adding a component to be searched for to the slide glass, staining the adhesive protein by an immunohistochemical staining method, and preparing a screening sample.
  • An evaluation step is provided for evaluating the effect of inhibiting the formation of the compact stratum corneum of the component to be searched by using the area of the stained portion in the analysis image as an index.
  • the cell surface has a negative charge (COO ⁇ ) due to the carboxy group of the phospholipid.
  • COO ⁇ negative charge due to the carboxy group of the phospholipid.
  • a screening control sample to which the component to be searched is not added is sampled.
  • the evaluation step compares the area of the stained portion of the screening sample with the area of the stained portion of the screening control sample and the area of the stained portion of the screening sample is small, the component to be searched is the adhesive protein. This is a step of evaluating that the amount is reduced. With such a form, it is possible to more accurately screen components that affect the increase or decrease of the adhesive protein.
  • a plurality of screening samples having the same type of the component to be searched but having different concentrations are prepared.
  • the evaluation step is a step of comparing the areas of the stained portions of the plurality of screening samples and evaluating the optimum concentration in the action of reducing the amount of adhesive protein. With such a form, not only screening of a component having an action of reducing the amount of adhesive protein but also its optimum concentration can be evaluated.
  • the adhesive protein is desmoglein1.
  • the slide glass is a glass slide NH 3 + is arranged on the surface.
  • NH 3 + is, COO cell surface - to form a ionic bond, it is possible to improve the adhesion to glass slide corneocytes.
  • the cleaning step is a step of immersing the slide glass in xylene.
  • the cleaning step is a step of immersing the slide glass in xylene.
  • the present invention that solves the above problems
  • a method for preparing a stratum corneum cell sample using the tape stripping method A transfer process that transfers the stratum corneum onto a glass slide whose surface is positively charged, It includes a cleaning step of cleaning the slide glass. By transferring the stratum corneum onto the slide glass whose surface is positively charged, it is possible to suppress the detachment of the stratum corneum cells from the slide glass in the subsequent washing step.
  • a glass slide said slides is NH 3 + is arranged on the surface.
  • a component that affects the increase or decrease of the adhesive protein can be screened more accurately than the conventional method. Further, according to the method for preparing stratum corneum cells of the present invention, exfoliation of stratum corneum cells on a slide glass can be suppressed.
  • the screening method of the present invention includes a stratum corneum cell sample preparation step S10, a screening sample preparation step S20, an image acquisition step S30, and an evaluation step S40.
  • Step S10 is a collection step S11 in which the stratum corneum cells are collected by the tape stripping method.
  • the tape stripping method is a general term for a method of peeling skin cells by attaching a pressure-sensitive adhesive typified by an adhesive tape to the skin and peeling it off.
  • the pressure-sensitive adhesive to be used is not particularly limited, but it is preferable to use cellophane tape from the viewpoint of convenience.
  • transfer step S12 is performed in which the stratum corneum cells collected in step S11 are transferred to a slide glass whose surface is positively charged.
  • step S12 by transferring the stratum corneum cells to the slide glass whose surface is positively charged, the stratum corneum cells are less likely to be detached from the slide glass due to electrostatic interaction.
  • slide glass whose surface is positively charged examples include slide glass whose surface is modified with polylysine, polyalkylamine, amino-modified silicone and the like.
  • a glass slide surface of the slide glass was sequence NH 3 +. NH 3 +, in order to form a carboxyl group and ionic phospholipid binding, corneocytes hardly more peeled from the slide glass,
  • the slides NH 3 + is arranged on the surface of the slide glass may be commercially available ones, for example, (manufactured by Matsunami Glass Industries, Ltd.) "PLL coated slide glass", “APS coated slide glass” (Matsunami Examples thereof include “MAS coated slide glass” (manufactured by Matsunami Glass Industry Co., Ltd.) and the like (manufactured by Matsunami Glass Industry Co., Ltd.).
  • a washing step S13 is performed to wash the slide glass to which the stratum corneum cells have been transferred.
  • the method for cleaning the slide glass is not particularly limited, and the slide glass can be cleaned using a shaking type or ultrasonic type washing machine. In the present invention, it is preferable to perform cleaning by immersing the slide glass in a cleaning agent containing an organic compound. With such a form, exfoliation of stratum corneum cells can be further suppressed.
  • the cleaning agent examples include organic solvents such as xylene, chloroform, and ethyl acetate, but it is preferable to use xylene that does not affect the amount of adherent protein in the stratum corneum cells.
  • the immersion time is preferably 3 hours or more, more preferably 6 hours or more, further preferably 12 hours or more, and particularly preferably 24 hours or more at room temperature. is there.
  • a screening sample preparation step S20 is then performed.
  • the component to be searched (hereinafter referred to as the component to be searched) is added to the stratum corneum cell sample.
  • the search target component is not particularly limited, but in view of applying a component having an action of reducing the amount of adhesive protein to humans, for example, a component applicable to cosmetics and a component applicable to food are selected as search target components.
  • a component applicable to cosmetics and a component applicable to food are selected as search target components.
  • Can be. Examples of such components include plant extracts.
  • the component to be searched is preferably diluted with phosphate buffered saline (PBS) according to the concentration to be screened.
  • PBS phosphate buffered saline
  • a screening control sample may be prepared without adding the component to be searched.
  • Such embodiments are suitable for screening the types of components that have the effect of reducing the amount of adhesive protein.
  • a plurality of screening samples having the same type of component to be searched but having different concentrations may be prepared. Such an embodiment is suitable for screening at an optimum concentration, which acts to reduce the amount of adhesive protein.
  • step S20 the stratum corneum cells to which the component to be searched are added are stained by an immunohistochemical staining method.
  • Staining by the immunohistochemical staining method can be performed by a conventional method, and a direct method, an indirect method and a sensitization method can be arbitrarily selected depending on the type of adhesive protein. Moreover, any blocking agent can be used if necessary.
  • the adhesive protein to be stained is not particularly limited as long as it is contained in the stratum corneum cells, but is preferably desmoglein 1.
  • Desmoglein 1 has a close correlation between proteolysis of desmosome components and cell desquamation, and screening of components that affect the increase or decrease in the amount of desmoglein 1 is very useful.
  • an image acquisition step S30 for taking an image of the stratum corneum cells stained with the adhesive protein is performed. Images are taken using a microscope.
  • the type of microscope is not particularly limited, and an appropriate microscope can be used depending on the immunohistochemical staining method performed in step S20. Examples of the microscope include an optical microscope, a confocal laser scanning microscope, an optical microscope, and an electron microscope.
  • a confocal laser scanning microscope it is preferable to use a confocal laser scanning microscope.
  • a confocal laser scanning microscope By using a confocal laser scanning microscope, it is possible to take a focused image without blurring the boundary between the stained part (adhesive protein) and the unstained part (part other than the adhesive protein, slide glass, etc.). , The accuracy of the screening method of the present invention using the stained portion as an index can be improved.
  • a confocal laser scanning microscope it is preferable to adopt a fluorescent antibody method as the immunohistochemical staining method.
  • the area value of the dyed part and / or the undyed part, or the ratio (%) of the area of the dyed part and / or the undyed part is calculated (hereinafter, the area value is simply referred to as the area). And both the area ratio).
  • image processing and area calculation described above can be performed using image analysis software.
  • image analysis software "Image J (National Institutes of Health, NIH)" and the like can be exemplified.
  • the area of the dyed portion is preferably the average value of the area calculated from a plurality of randomly selected ranges. By using the average value of the areas of a plurality of locations as an index, more accurate screening can be performed.
  • evaluation step S40 is performed to evaluate the effect of the component to be searched for on the increase or decrease of the adhesive protein.
  • FIG. 3 shows a flowchart showing step S40 in detail.
  • the evaluation step in one embodiment in which the area of the stained portion of the screening sample and the area of the stained portion of the screening control sample are compared with reference to FIG. 3 will be described.
  • step S41 the area At of the stained portion of the screening sample and the area Ac of the stained portion of the screening sample are compared.
  • the comparison of the areas may be a comparison of the absolute values of the areas, or may be a comparison of the ratio of the areas when the area of the screening control sample is 100%.
  • step S42 the search target component added to the screening sample is evaluated as a component having an action of reducing the amount of adhesive protein.
  • the process proceeds to step S43, and the search target component added to the screening sample is the adhesive protein. It is evaluated as a component having an action of reducing the amount.
  • the evaluation step in the screening method of the present invention uses the area of the stained portion of the screening sample as an index to evaluate the effect on the increase or decrease of the adhesive protein. If so, there is no particular limitation.
  • step S41 when it is desired to screen a component that increases the amount of adhesive protein, it is determined in step S41 whether "Area At> Area Ac", and if the result is "yes", in step S42. It may be evaluated as "a component having an action of increasing the amount of adhesive protein". Further, in step S42, it may be evaluated as "a component having an action of reducing the amount of adhesive protein", and in step S43, it may be evaluated as "a component having an action of increasing the amount of adhesive protein”. ..
  • step S20 there may be a step of preparing a plurality of screening samples having different concentrations of the components to be searched and evaluating them.
  • the areas of a plurality of screening samples are compared, and when there is a specific concentration that becomes a threshold value at which a remarkable effect is exhibited in the action of reducing or increasing the amount of adhesive protein.
  • the component to be searched has an action of significantly reducing or increasing the amount of adhesive protein that is remarkable above a specific concentration.”
  • the concentration is above a specific concentration
  • the screening method of the present invention is preferably used for screening components used for improving and / or preventing sensitive skin.
  • sensitive skin refers to a skin type having reduced resistance to external stimuli (dryness, ultraviolet rays, application of compounds) (decreased skin stimulus value).
  • sensitive skin in the present specification refers to the skin type raised in (1) and / or (2).
  • "for improvement and / or prevention of sensitive skin” in the present specification means improvement of skin quality in which external stimulus resistance is reduced (skin stimulus threshold is lowered) and / or external stimulus resistance in skin quality. Refers to the prevention of a decrease in skin irritation (decrease in skin irritation threshold).
  • the screening method of the present invention is preferably used for screening a component having an intercellular adhesion inhibitory effect on human epidermal keratinocytes.
  • cell adhesion refers to dysfunction of stratum corneum adhesion due to an increase in desmosome protein (protein group containing desmoglein) (progression of stratification of stratum corneum due to enhanced cell adhesion).
  • desmosome protein protein group containing desmoglein
  • the concept of "inhibition of cell-cell adhesion” includes any of prevention or improvement of cell-cell adhesion.
  • the screening method of the present invention can be used for screening components having a preventive or ameliorating effect on exfoliation of stratum corneum.
  • exfoliation of stratum corneum means exfoliation from the skin surface in a state where a plurality of stratum corneum are overlapped. Whether or not the skin is exfoliated from the stratum corneum can be confirmed by collecting the stratum corneum by the tape stripping method, staining the collected stratum corneum, and observing it.
  • the component having an action of reducing desmoglein is a component having an action of improving the lightness of the skin. That is, the screening method of the present invention can be used for screening components having an effect of improving skin brightness.
  • "improvement of lightness of skin” means that the difference in lightness between normal skin and skin whose lightness is reduced due to the excessive presence of desmoglein is reduced.
  • a component having a desmoglein reducing action has an action of improving a more transparent skin condition. That is, the screening method of the present invention can be used for screening components having an effect of improving the transparency of the skin.
  • a large amount of desmoglein 1 is present in a place where the hardness of the skin is high, and it is considered that the factor of the hardness of the skin is due to the stratification of the stratum corneum.
  • reducing desmoglein can have the effect of softening the skin. That is, the screening method of the present invention can be used for screening components having a skin softening effect.
  • the screening method of the present invention can be used for screening a component having an action of more efficiently permeating a medicinal component such as a whitening agent into the skin.
  • Example 1 ⁇ Preparation process of stratum corneum cell sample> From the outside of the human forearm, the stratum corneum collected with cellophane tape was transferred to a slide glass (“MAS coat”, manufactured by Matsunami Glass Industry Co., Ltd.), and the slide glass was immersed in xylene overnight. The slide glass was then washed twice with xylene for 10 minutes and then with PBS.
  • MAS coat manufactured by Matsunami Glass Industry Co., Ltd.
  • ⁇ Preparation of search target components Wild thyme extract (above-ground extract of Wild Time, T. serpylum, manufactured by Ichimaru Falcos) and spiny extract (fruit extract of ROSA roxburghii, manufactured by Maruzen Pharmaceuticals Co., Ltd.)
  • the components to be searched were obtained by diluting with PBS so that the concentrations were 0.1% by mass, 0.05% by mass, 0.025% by mass, and 0.0125% by mass, respectively.
  • ⁇ Preparation of screening sample A sample was obtained by immersing the stratum corneum cells in the component to be searched overnight. In addition, as a control sample, stratum corneum cells not immersed in the component to be searched were prepared.
  • the screening sample in which the concentration of the added wild thyme extract was 0.1% by mass was higher than that in the screening sample in which the concentrations were 0.05, 0.025, and 0.0125% by mass. It can be seen that the area is reduced by about 2.5 to 2.8 times.
  • the screening sample with a concentration of 0.1% by weight of the added spiny extract was compared to the screening sample with concentrations of 0.05, 0.025, and 0.0125% by weight of the stained portion. It can be seen that the area is reduced by 7.6 to 8.0 times.
  • the wild thyme extract and the spiny extract have a remarkable effect of reducing the amount of desmoglein 1 at a concentration of 0.1% by mass or more.
  • Example 2 ⁇ Preparation process of stratum corneum cell sample> A stratum corneum cell sample was prepared by the same method as in Example 1.
  • the Marsh mallow extract extract of the root of Marsh mallow (Althaea Officinalis) manufactured by jurlique) was diluted with PBS to a concentration of 0.25% by mass to obtain a component to be searched.
  • ⁇ Preparation of screening sample> A sample was obtained by immersing the stratum corneum cells in the component to be searched overnight. In addition, as a control sample, stratum corneum cells not immersed in the search target component were prepared.
  • a screening sample and a screening control sample were obtained by the same method as in Example 1.
  • Example 3 The test results supporting the relationship between the difference in skin brightness and the abundance of desmoglein 1 and the relationship between the abundance of desmoglein 1 and the hardness of the skin are shown below.
  • Subjects and measurement sites In this example, 20 women aged 40 to 52 years were used as subjects. Then, in this example, a part of the subject's face where it was confirmed that the brightness was visually reduced (blackish part, see part A in FIG. 13) and a part in a normal state (see part B in FIG. 13). ) was selected as the measurement site.
  • the part where it was confirmed that the brightness was visually reduced was compared with the part in the normal state (see part B in FIG. 13).
  • the part where it was confirmed that the brightness was visually reduced was compared with the part in the normal state (see part B in FIG. 13).
  • the part where it was confirmed that the brightness was visually reduced was significant compared to the part in the normal state (see part B in FIG. 13). Showed a low value. That is, the part where it was confirmed that the brightness was visually reduced (blackish part, see part A in FIG. 13) was harder than the part in the normal state (see part B in FIG. 13). Was confirmed.
  • the slide glass to which the stratum corneum cells were attached was immersed in xylene overnight. After immersion, it was washed and air-dried, and then allowed to stand in a 4% paraformaldehyde / phosphate buffer solution at room temperature for 15 minutes.
  • the cells were framed with a liquid blocker (manufactured by Daido Sangyo Co., Ltd.), the primary antibody was added, and the mixture was allowed to stand in a wet box at room temperature for 2 hours.
  • Anti Desmoglein 1, Mouse Mono (Dsg1 P23) Progen, 651110 undiluted solution (IgG concentration: 10-15 g / mL) was used as the primary antibody.
  • the part where it was visually confirmed that the brightness was reduced is the part in the normal state (see part B in FIG. 13).
  • the value was significantly higher than that of. That is, the part where the brightness was visually confirmed to be reduced (blackish part, see part A in FIG. 13) was desmoglein as compared with the part in the normal state (see part B in FIG. 13). It was confirmed that the amount of 1 (Dsg1) was large.
  • the active ingredient of the present invention exerts a desmoglein reducing action.
  • the active ingredient of the present invention exerts an effect of improving the brightness of the skin by reducing desmoglein 1.
  • the area of the fluorescent part was calculated with the area of the entire analysis image as 100%. The results are shown in FIGS. 14 and 15. In FIG. 15, * is p ⁇ 0.05.
  • the optical microscope image of the stratum corneum cells before use of Production Example 1 has a plurality of dark-colored portions.
  • This dark part is the part where the stratum corneum cells have been exfoliated by the tape stripping method.
  • most of the stratum corneum cell samples collected after using Production Example 1 for 10 days are lightly stained as compared with the stratum corneum cell samples collected before use.
  • This pale part means that only one layer of stratum corneum cells has been exfoliated, that is, no multi-layer exfoliation has occurred.
  • the multi-layer peeling was improved by using the cosmetic product of Production Example 1 containing a component having a desmoglein reducing effect.
  • Example 5 Hereinafter, the desmoglein-reducing ability of a composition containing a component having a desmoglein-reducing effect was investigated.
  • composition A cosmetic (milky lotion) containing a wild thyme extract was prepared according to the formulation shown in Table 4 below. That is, each of the components (a), (b), and (c) is heated to 70 ° C., (c) is added to (b) to neutralize, and (a) is gradually added while stirring this. After emulsification and homogenization using a homogenizer, the mixture was cooled with stirring to obtain the emulsion of Production Example 2.
  • the area of the fluorescent part was selected with the area of the entire analysis image as 100%.
  • the results are shown in Tables 5, 6, and 17 (representative), 18 and 19.
  • the data after 12 weeks of using the emulsion the data of 80 of the 93 spots were aggregated and compared with the data before using the emulsion at the same site. Further, in FIG. 18, ** is p ⁇ 0.01, and in FIG. 19, *** is p ⁇ 0.001.
  • the present invention can be applied to analytical methods related to the amount of adhesive protein.

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Abstract

The present invention provides a method for screening a component that affects fluctuation of desmoglein and other adhesion proteins present in stratum corneum cells, the method being more accurate than conventional methods.　A method for screening a component that affects fluctuation of adhesion proteins in stratum corneum cells, wherein the screening method comprises: a harvesting step for harvesting stratum corneum cells using a deep stripping technique; a transfer step for transferring the stratum corneum to a slide the surface of which has been positively charged; a cleansing step for cleansing the slide; an adjustment step for adding a sought component to the slide, dyeing adhesion proteins through an immune tissue dyeing technique, and adjusting a screening specimen; an image acquisition step for photographing a discretionary location on the screening specimen and obtaining an image for analysis; and an evaluation step for evaluating the effect of the sought component on fluctuation of the adhesion proteins, with the area of a dyed portion in the image for analysis being used as an indicator.

Description

角層細胞間の接着タンパク質の増減に影響を与える成分のスクリーニング方法Screening method for components that affect the increase or decrease of adhesive proteins between stratum corneum cells
　本発明は、角層細胞間の接着タンパク質の増減に影響を与える成分のスクリーニング方法、及び当該スクリーニング方法に適した角層細胞試料の調製方法に関する。 The present invention relates to a method for screening components that affect the increase or decrease of adhesive proteins between stratum corneum cells, and a method for preparing a stratum corneum cell sample suitable for the screening method.
　角化細胞（ケラチノサイト）の接着装置として代表的なものにデスモソームがある。デスモソームは、表皮細胞間及び角層細胞間の接着に関与している構造物であり、デスモソームを介する表皮細胞同士の接着はケラチン線維からなる細胞骨格と連携し、上皮組織における強固な構築が維持されるものであることが知られている。 Desmosome is a typical adhesive device for keratinocytes (keratinocytes). Desmosomes are structures involved in the adhesion between epidermal cells and stratum corneum cells, and the adhesion between epidermal cells via desmosomes cooperates with the cytoskeleton consisting of keratin fibers to maintain a strong structure in epithelial tissue. It is known to be done.
　肌荒れ、日焼けや乾燥により生じる角層の落屑等の肌状態の悪化に関し、デスモゾームタンパク質の増加が原因の一つとして知られている。
　デスモゾームタンパク質の量をコントロールする方法として、角層に蓄積したデスモゾームタンパク質のプロテアーゼにより分解し、ニキビ、フケ、落屑を改善する方法がこれまでに報告されている（特許文献１）。 It is known that an increase in desmosome protein is one of the causes of deterioration of skin condition such as desquamation of the stratum corneum caused by rough skin, sunburn and dryness.
As a method for controlling the amount of desmosome protein, a method for improving acne, dandruff, and desquamation by degrading with a protease of desmosome protein accumulated in the stratum corneum has been reported so far (Patent Document 1).
　このデスモソームの構成タンパク質は、デスモグレイン１、２、３、４、デスモコリン１、２、３、及びデスモプラキン１、２等が知られている。
　このデスモソームの構成タンパクの中でも、特に接着タンパク質のデスモグレイン１が、デスモソーム成分のタンパク分解と細胞剥離との間に密接な相関関係がある。このため、角層のデスモグレインの存在状態を確認することは、デスモソーム機能を評価することでもあり、皮膚の状態を評価する上で極めて重要である。 Desmoglein 1, 2, 3, 4, desmocollin 1, 2, 3, and desmoplakin 1, 2, etc. are known as constituent proteins of this desmosome.
Among the constituent proteins of this desmosome, desmoglein 1, which is an adhesive protein, has a close correlation between proteolysis of desmosome components and cell detachment. Therefore, confirming the presence state of desmoglein in the stratum corneum is also an evaluation of desmosome function, and is extremely important in evaluating the state of the skin.
特表平７－５０５３８３号公報Special Table No. 7-505383
　上述したことから、デスモソーム、又はデスモソームを構成する接着タンパク質の量を減少する成分の探索が求められている。
　このような成分のスクリーニング方法として、テープストリッピング法を用いた角層細胞の採取、及びスライドガラスへの転写により、角層細胞試料を調製し、免疫組織染色法により接着タンパク質を染色し、任意の顕微鏡で観察する手法が存在する。 From the above, it is required to search for desmosomes or components that reduce the amount of adhesive proteins constituting the desmosomes.
As a screening method for such components, a stratum corneum cell sample is prepared by collecting stratum corneum cells using a tape stripping method and transferring to a slide glass, and an adhesive protein is stained by an immune tissue staining method, and any There is a method of observing with a microscope.
　ところで、テープストリッピング法を用いた角層細胞の転写は、スライドガラスに粘着テープの成分が残り、これが染色されることで接着タンパク質と区別できなくなる恐れがあるため、スライドガラスを洗浄する必要がある。
　しかし、スライドガラスを洗浄すると、転写した角層細胞まで剥離してしまい、角層細胞中に存在する接着タンパク質の存在量を指標とした評価が困難であり、十分なスクリーニング方法であるといえなかった。 By the way, in the transcription of stratum corneum cells using the tape stripping method, the components of the adhesive tape remain on the slide glass, which may be stained and indistinguishable from the adhesive protein, so it is necessary to wash the slide glass. ..
However, when the slide glass is washed, even the transferred stratum corneum cells are exfoliated, and it is difficult to evaluate using the abundance of adhesive proteins present in the stratum corneum cells as an index, so it cannot be said that this is a sufficient screening method. It was.
　本発明の課題は、従来の方法と比して正確な、デスモグレインをはじめとする角層細胞間に存在する接着タンパク質の増減に影響を与える成分のスクリーニング方法を提供することにある。
　また、本発明のさらなる課題は、上述したスクリーニング方法において、より正確に接着タンパク質の存在量を評価するための角層細胞試料の調製方法を提供することにある。 An object of the present invention is to provide a method for screening components that affect the increase or decrease of adhesive proteins existing between stratum corneum cells such as desmoglein, which is more accurate than the conventional method.
A further object of the present invention is to provide a method for preparing a stratum corneum cell sample for more accurately evaluating the abundance of an adhesive protein in the above-mentioned screening method.
　前記課題を解決する本発明は、
　角層細胞間の接着タンパク質の増減に影響を与える成分のスクリーニング方法であって、
　テープストリッピング法を用いて、角層細胞を採取する採取工程と、
　表面が正電荷に帯電したスライドガラス上に、角層を転写する転写工程と、
　前記スライドガラスを洗浄する洗浄工程と、
　前記スライドガラスに探索対象成分を添加し、免疫組織染色法により、接着タンパク質を染色し、スクリーニング試料を調製する調製工程と、
　前記スクリーニング試料の任意の箇所を撮影し、解析用画像を得る画像取得工程と、
　前記解析用画像における染色部分の面積を指標として、前記探索対象成分のコンパクト角層の形成阻害作用を評価する評価工程を備える。 The present invention that solves the above problems
A screening method for components that affect the increase or decrease of adhesive proteins between stratum corneum cells.
A collection process for collecting stratum corneum cells using the tape stripping method,
A transfer process that transfers the stratum corneum onto a glass slide whose surface is positively charged,
A cleaning process for cleaning the slide glass and
A preparation step of adding a component to be searched for to the slide glass, staining the adhesive protein by an immunohistochemical staining method, and preparing a screening sample.
An image acquisition step of photographing an arbitrary part of the screening sample to obtain an image for analysis, and
An evaluation step is provided for evaluating the effect of inhibiting the formation of the compact stratum corneum of the component to be searched by using the area of the stained portion in the analysis image as an index.
　細胞表面は、リン脂質のカルボキシ基により、負電荷（ＣＯＯ－）を有することが知られている。
　本発明のスクリーニング方法によれば、表面が正電荷に帯電したスライドガラス上に、角層細胞を転写することで、洗浄による角層細胞の剥離を防止することができ、より正確に角層細胞中の接着タンパク質の存在量を評価することができる。 It is known that the cell surface has a negative charge (COO − ) due to the carboxy group of the phospholipid.
According to the screening method of the present invention, by transferring the stratum corneum cells onto a slide glass whose surface is positively charged, it is possible to prevent the stratum corneum cells from being detached by washing, and more accurately. The abundance of adhesive proteins in the cells can be evaluated.
　本発明の好ましい形態では、
　前記調製工程において、前記探索対象成分を添加しないスクリーニング対照試料を試料し、
　前記評価工程が、前記スクリーニング試料の染色部分の面積と、前記スクリーニング対照試料の染色部分の面積とを比較して、前記スクリーニング試料の染色部分の面積が小さい場合、前記探索対象成分は接着タンパク質の量を減少する作用を有すると評価する工程である。
　このような形態とすることで、より正確に接着タンパク質の増減に影響を与える成分をスクリーニングすることができる。 In a preferred embodiment of the invention
In the preparation step, a screening control sample to which the component to be searched is not added is sampled.
When the evaluation step compares the area of the stained portion of the screening sample with the area of the stained portion of the screening control sample and the area of the stained portion of the screening sample is small, the component to be searched is the adhesive protein. This is a step of evaluating that the amount is reduced.
With such a form, it is possible to more accurately screen components that affect the increase or decrease of the adhesive protein.
　本発明の好ましい形態では、
　前記調製工程において、前記探索対象成分の種類が同一であって、濃度が異なる複数のスクリーニング試料を調製し、
　前記評価工程が、複数の前記スクリーニング試料の染色部分の面積を比較して、接着タンパク質の量を減少する作用における最適濃度を評価する工程である。
　このような形態とすることで、接着タンパク質の量を減少する作用を有する成分のスクリーニングのみならず、その最適な濃度を評価することができる。 In a preferred embodiment of the invention
In the preparation step, a plurality of screening samples having the same type of the component to be searched but having different concentrations are prepared.
The evaluation step is a step of comparing the areas of the stained portions of the plurality of screening samples and evaluating the optimum concentration in the action of reducing the amount of adhesive protein.
With such a form, not only screening of a component having an action of reducing the amount of adhesive protein but also its optimum concentration can be evaluated.
　本発明の好ましい形態では、前記接着タンパク質が、デスモグレイン１（ｄｅｓｍｏｇｌｅｉｎ１）である。 In the preferred embodiment of the present invention, the adhesive protein is desmoglein1.
　本発明の好ましい形態では、前記スライドガラスが、表面にＮＨ３ ＋が配列されたスライドガラスである。
　ＮＨ３ ＋は、細胞表面のＣＯＯ－とイオン結合を形成するため、角層細胞のスライドガラスへの接着性を向上させることができる。 In a preferred form of the present invention, the slide glass is a glass slide NH 3 + is arranged on the surface.
NH 3 + is, COO cell surface - to form a ionic bond, it is possible to improve the adhesion to glass slide corneocytes.
　本発明の好ましい形態では、前記洗浄工程が、前記スライドガラスをキシレンに浸漬する工程である。
　スライドガラスをキシレンに浸漬することにより洗浄することで、角層細胞の剥離をより抑制することができる。 In a preferred embodiment of the present invention, the cleaning step is a step of immersing the slide glass in xylene.
By washing the slide glass by immersing it in xylene, the detachment of stratum corneum cells can be further suppressed.
　また、前記課題を解決する本発明は、
　テープストリッピング法を用いた角層細胞試料の調製方法であって、
　表面が正電荷に帯電したスライドガラス上に、角層を転写する転写工程と、
　前記スライドガラスを洗浄する洗浄工程と、を備える。
　表面が正電荷に帯電したスライドガラス上に、角層を転写することで、その後の洗浄工程における、スライドガラスからの角層細胞の剥離を抑制することができる。 In addition, the present invention that solves the above problems
A method for preparing a stratum corneum cell sample using the tape stripping method.
A transfer process that transfers the stratum corneum onto a glass slide whose surface is positively charged,
It includes a cleaning step of cleaning the slide glass.
By transferring the stratum corneum onto the slide glass whose surface is positively charged, it is possible to suppress the detachment of the stratum corneum cells from the slide glass in the subsequent washing step.
　本発明の好ましい形態では、前記スライドガラスが表面にＮＨ３ ＋が配列されたスライドガラスである。 In a preferred form of the present invention, a glass slide said slides is NH 3 + is arranged on the surface.
　本発明のスクリーニング方法によれば、従来の方法と比してより正確に、接着タンパク質の増減に影響を与える成分をスクリーニングすることができる。
　また、本発明の角層細胞の調製方法によれば、スライドガラス上の角層細胞の剥離を抑制することができる。 According to the screening method of the present invention, a component that affects the increase or decrease of the adhesive protein can be screened more accurately than the conventional method.
Further, according to the method for preparing stratum corneum cells of the present invention, exfoliation of stratum corneum cells on a slide glass can be suppressed.
本実施形態のスクリーニング方法を示すフローチャートである。It is a flowchart which shows the screening method of this embodiment. 本実施形態における角層細胞試料の調製工程を示すフローチャートである。It is a flowchart which shows the preparation process of the stratum corneum cell sample in this embodiment. 本実施形態における評価工程を示すフローチャートである。It is a flowchart which shows the evaluation process in this embodiment. 角層細胞試料にワイルドタイム抽出物、トゲナシ抽出物を探索対象成分として添加したスクリーニング試料、及びスクリーニング対照試料の顕微鏡画像である。It is a microscopic image of a screening sample in which a wild thyme extract and a spiny extract were added as search target components to a stratum corneum cell sample, and a screening control sample. スクリーニング対照試料の染色部分の面積を１００％とした場合における、スクリーニング試料の染色部分の面積比を表わすグラフである。3 is a graph showing the area ratio of the stained portion of the screening sample when the area of the stained portion of the screening control sample is 100%. 実施例２における、デスモグレイン１量の測定結果を示す図面（代表）である。It is a drawing (representative) which shows the measurement result of 1 amount of desmoglein in Example 2. 実施例２における、デスモグレイン１量の測定結果を示すグラフである。It is a graph which shows the measurement result of 1 amount of desmoglein in Example 2. 実施例３における、L＊値の測定結果を示すグラフである。It is a graph which shows the measurement result of the L * value in Example 3. 実施例３における、a＊値の測定結果を示すグラフである。It is a graph which shows the measurement result of a * value in Example 3. 実施例３における、b＊値の測定結果を示すグラフである。It is a graph which shows the measurement result of the b * value in Example 3. 実施例３における、硬度の測定結果を示すグラフである。It is a graph which shows the measurement result of hardness in Example 3. 実施例３における、デスモグレイン１量の測定結果を示すグラフである。It is a graph which shows the measurement result of 1 amount of desmoglein in Example 3. 実施例３における、デスモグレイン１量の測定結果を示す図（代表）である。It is a figure (representative) which shows the measurement result of 1 amount of desmoglein in Example 3. 実施例４における、デスモグレイン１量の測定結果を示す図（代表）である。It is a figure (representative) which shows the measurement result of 1 amount of desmoglein in Example 4. 実施例４における、デスモグレイン１量の測定結果を示すグラフである。It is a graph which shows the measurement result of 1 amount of desmoglein in Example 4. 実施例４における、角層細胞の剥離の様子を観察した光学顕微鏡像である。It is an optical microscope image which observed the state of the exfoliation of the stratum corneum cell in Example 4. 実施例５における、デスモグレイン１量の測定結果を示す図（代表）である。It is a figure (representative) which shows the measurement result of 1 amount of desmoglein in Example 5. 実施例５における、デスモグレイン１量の測定結果を示すグラフ（６週間）である。It is a graph (6 weeks) which shows the measurement result of 1 amount of desmoglein in Example 5. 実施例５における、デスモグレイン１量の測定結果を示すグラフ（１２週間）である。It is a graph (12 weeks) which shows the measurement result of 1 amount of desmoglein in Example 5.
　以下、図１～３のフローチャートを参考にして、本発明のスクリーニング方法について詳述する。 Hereinafter, the screening method of the present invention will be described in detail with reference to the flowcharts of FIGS.
　本発明のスクリーニング方法は、角層細胞試料調製ステップＳ１０、スクリーニング試料調製ステップＳ２０、画像取得ステップＳ３０、及び評価ステップＳ４０を備える。 The screening method of the present invention includes a stratum corneum cell sample preparation step S10, a screening sample preparation step S20, an image acquisition step S30, and an evaluation step S40.
（１）角層細胞試料調製工程
　図２に、ステップＳ１０を詳細に表したフローチャートを示す。
　ステップＳ１０は、テープストリッピング法により角層細胞を採取する採取ステップＳ１１を行う。
　テープストリッピング法とは、粘着テープに代表される感圧接着剤を肌に貼り付け、剥がすことで肌の細胞を剥離する方法の総称である。
　使用する感圧接着剤は特に限定されないが、簡便性の観点からセロハンテープを用いることが好ましい。 (1) Horn layer cell sample preparation step FIG. 2 shows a flowchart showing step S10 in detail.
Step S10 is a collection step S11 in which the stratum corneum cells are collected by the tape stripping method.
The tape stripping method is a general term for a method of peeling skin cells by attaching a pressure-sensitive adhesive typified by an adhesive tape to the skin and peeling it off.
The pressure-sensitive adhesive to be used is not particularly limited, but it is preferable to use cellophane tape from the viewpoint of convenience.
　次いで、ステップＳ１１で採取した角層細胞を、表面が正電荷に帯電したスライドガラスに転写する転写ステップＳ１２を行う。 Next, transfer step S12 is performed in which the stratum corneum cells collected in step S11 are transferred to a slide glass whose surface is positively charged.
　角層細胞等のヒト細胞の表面は、脂質二重膜を形成するリン脂質のカルボキシ基（ＣＯＯ－）の影響で、負電荷を帯びている。
　ステップＳ１２において、表面が正電荷に帯電したスライドガラスに角層細胞を転写することによって、静電的相互作用により角層細胞がスライドガラスから剥離し難くなる。 The surface of human cells such as stratum corneum cells is negatively charged due to the influence of the carboxy group (COO − ) of phospholipids that form a lipid bilayer.
In step S12, by transferring the stratum corneum cells to the slide glass whose surface is positively charged, the stratum corneum cells are less likely to be detached from the slide glass due to electrostatic interaction.
　表面が正電荷に帯電したスライドガラスとしては、スライドガラス表面をポリリジン、ポリアルキルアミン、及びアミノ変性シリコーン等で修飾したスライドガラスが例示できる。
　本発明においては、スライドガラスの表面がＮＨ３ ＋が配列したスライドガラスを用いることが好ましい。
　ＮＨ３ ＋は、リン脂質のカルボキシ基とイオン結合を形成するため、角層細胞がスライドガラスからより剥離し難くなる、 Examples of the slide glass whose surface is positively charged include slide glass whose surface is modified with polylysine, polyalkylamine, amino-modified silicone and the like.
In the present invention, it is preferable to use a glass slide surface of the slide glass was sequence NH 3 +.
NH 3 +, in order to form a carboxyl group and ionic phospholipid binding, corneocytes hardly more peeled from the slide glass,
　スライドガラスの表面にＮＨ３ ＋が配列したスライドガラスとしては、市販の物を用いることができ、例えば、「ＰＬＬコートスライドグラス」（松浪硝子工業株式会社製）、「ＡＰＳコートスライドグラス」（松浪硝子工業株式会社製）、及び「ＭＡＳコートスライドグラス」（松浪硝子工業株式会社製）等が例示できる。 The slides NH 3 + is arranged on the surface of the slide glass may be commercially available ones, for example, (manufactured by Matsunami Glass Industries, Ltd.) "PLL coated slide glass", "APS coated slide glass" (Matsunami Examples thereof include "MAS coated slide glass" (manufactured by Matsunami Glass Industry Co., Ltd.) and the like (manufactured by Matsunami Glass Industry Co., Ltd.).
　次いで、角層細胞を転写したスライドガラスを洗浄する洗浄ステップＳ１３を行う。
　スライドガラスの洗浄方法は、特に限定されず、振とう式、及び超音波式の洗浄機を用いて洗浄することができる。
　本発明においては、有機化合物を含む洗浄剤にスライドガラスを浸漬することで洗浄することが好ましい。
　このような形態とすることで、より角層細胞の剥離を抑制することができる。 Next, a washing step S13 is performed to wash the slide glass to which the stratum corneum cells have been transferred.
The method for cleaning the slide glass is not particularly limited, and the slide glass can be cleaned using a shaking type or ultrasonic type washing machine.
In the present invention, it is preferable to perform cleaning by immersing the slide glass in a cleaning agent containing an organic compound.
With such a form, exfoliation of stratum corneum cells can be further suppressed.
　洗浄剤としては、キシレン、クロロホルム、酢酸エチル等の有機溶剤が例示できるが、角層細胞の接着タンパク質の量に影響を及ぼさないキシレンを用いることが好ましい。 Examples of the cleaning agent include organic solvents such as xylene, chloroform, and ethyl acetate, but it is preferable to use xylene that does not affect the amount of adherent protein in the stratum corneum cells.
　浸漬による洗浄を行う場合、その浸漬時間は、室温であれば、好ましくは３時間以上であり、より好ましくは６時間以上であり、さらに好ましくは１２時間以上であり、特に好ましくは２４時間以上である。 When washing by immersion, the immersion time is preferably 3 hours or more, more preferably 6 hours or more, further preferably 12 hours or more, and particularly preferably 24 hours or more at room temperature. is there.
　上述したステップを得て、角層細胞試料を調製したら、次いでスクリーニング試料調製ステップＳ２０を行う。
　ステップＳ２０は、探索対象となる成分（以下、探索対象成分という）を、角層細胞試料に添加する。 After obtaining the above-mentioned steps and preparing a stratum corneum cell sample, a screening sample preparation step S20 is then performed.
In step S20, the component to be searched (hereinafter referred to as the component to be searched) is added to the stratum corneum cell sample.
　探索対象成分は特に限定されないが、接着タンパク質の量を減少する作用を有する成分をヒトに適用することに鑑みれば、例えば、化粧品に適用可能な成分、及び食品に適用可能な成分を探索対象成分とすることができる。
　このような成分として、例えば植物抽出物が例示できる。 The search target component is not particularly limited, but in view of applying a component having an action of reducing the amount of adhesive protein to humans, for example, a component applicable to cosmetics and a component applicable to food are selected as search target components. Can be.
Examples of such components include plant extracts.
　探索対象成分は、スクリーニングを行う濃度に応じて、リン酸緩衝生理食塩水（ＰＢＳ）で希釈することが好ましい。 The component to be searched is preferably diluted with phosphate buffered saline (PBS) according to the concentration to be screened.
　本発明の一実施形態においては、探索対象成分を添加しない、スクリーニング対照試料を調製してもよい。
　このような実施形態は、接着タンパク質の量を減少する作用を有する成分の種類のスクリーニングに適している。 In one embodiment of the present invention, a screening control sample may be prepared without adding the component to be searched.
Such embodiments are suitable for screening the types of components that have the effect of reducing the amount of adhesive protein.
　本発明の一実施形態においては、探索対象成分の種類が同一であって、濃度が異なる複数のスクリーニング試料を調製してもよい。
　このような実施形態は、接着タンパク質の量を減少する作用を奏する、最適な濃度のスクリーニングに適している。 In one embodiment of the present invention, a plurality of screening samples having the same type of component to be searched but having different concentrations may be prepared.
Such an embodiment is suitable for screening at an optimum concentration, which acts to reduce the amount of adhesive protein.
　また、ステップＳ２０において、探索対象成分を添加した角層細胞を、免疫組織染色法により染色する。
　免疫組織染色法による染色は、常法により行うことができ、接着タンパク質の種類に応じて、直接法、間接法及び増感法を任意に選択することができる。
　また、必要に応じて、任意のブロッキング剤を用いることができる。 Further, in step S20, the stratum corneum cells to which the component to be searched are added are stained by an immunohistochemical staining method.
Staining by the immunohistochemical staining method can be performed by a conventional method, and a direct method, an indirect method and a sensitization method can be arbitrarily selected depending on the type of adhesive protein.
Moreover, any blocking agent can be used if necessary.
　染色する対象となる接着タンパク質は、角層細胞に含まれるものであれば特に限定されないが、好ましくはデスモグレイン１である。
　デスモグレイン１は、デスモソーム成分のタンパク分解と細胞剥離との間に密接な相関関係があり、その量の増減に影響を与える成分のスクリーニングは、非常に有用である。 The adhesive protein to be stained is not particularly limited as long as it is contained in the stratum corneum cells, but is preferably desmoglein 1.
Desmoglein 1 has a close correlation between proteolysis of desmosome components and cell desquamation, and screening of components that affect the increase or decrease in the amount of desmoglein 1 is very useful.
　次いで、接着タンパク質を染色した角層細胞の画像を撮影する画像取得ステップＳ３０を行う。
　画像の撮影は、顕微鏡を用いて行われる。顕微鏡の種類は特に限定されず、ステップＳ２０で行った免疫組織染色法に応じて、適当な顕微鏡を用いることができる。
　顕微鏡としては、光学顕微鏡、共焦点レーザー顕微鏡、光学顕微鏡、電子顕微鏡等が挙げられる。 Next, an image acquisition step S30 for taking an image of the stratum corneum cells stained with the adhesive protein is performed.
Images are taken using a microscope. The type of microscope is not particularly limited, and an appropriate microscope can be used depending on the immunohistochemical staining method performed in step S20.
Examples of the microscope include an optical microscope, a confocal laser scanning microscope, an optical microscope, and an electron microscope.
　本発明においては、共焦点レーザー顕微鏡を用いることが好ましい。
　共焦点レーザー顕微鏡を用いることで、染色部分（接着タンパク質）と非染色部分（接着タンパク質以外の部分、スライドガラス等）との境界がぼけずに、ピントがあった画像を撮影することができるため、染色部分を指標とした本発明のスクリーニング方法の精度を上げることができる。
　なお、共焦点レーザー顕微鏡を用いる場合には、前記免疫組織染色法として、蛍光抗体法を採用することが好ましい。 In the present invention, it is preferable to use a confocal laser scanning microscope.
By using a confocal laser scanning microscope, it is possible to take a focused image without blurring the boundary between the stained part (adhesive protein) and the unstained part (part other than the adhesive protein, slide glass, etc.). , The accuracy of the screening method of the present invention using the stained portion as an index can be improved.
When a confocal laser scanning microscope is used, it is preferable to adopt a fluorescent antibody method as the immunohistochemical staining method.
　次いで、撮影した画像から、角層細胞が転写された部分内において、無作為に選択した範囲を拡大し、白黒画像（二値画像）に変換する。
　得られた画像から、染色部分、及び／又は非染色部分の面積値、あるいは染色部分及び／又は非染色部分の面積の割合（％）を算出する（以下、単に面積というときは、面積値、及び面積の割合の両方を含む）。 Then, from the captured image, a randomly selected range is enlarged in the portion where the stratum corneum cells are transferred and converted into a black-and-white image (binary image).
From the obtained image, the area value of the dyed part and / or the undyed part, or the ratio (%) of the area of the dyed part and / or the undyed part is calculated (hereinafter, the area value is simply referred to as the area). And both the area ratio).
　上述した画像処理、及び面積の算出については、画像解析ソフトを用いて行うことができる。
　画像解析ソフトとしては、「Ｉｍａｇｅ　Ｊ（アメリカ国立衛生研究所、ＮＩＨ）」等が例示できる。 The image processing and area calculation described above can be performed using image analysis software.
As the image analysis software, "Image J (National Institutes of Health, NIH)" and the like can be exemplified.
　染色部分の面積は、複数の無作為に選択した範囲から算出した面積の平均値とすることが好ましい。
　複数箇所の面積の平均値を指標とすることで、より正確なスクリーニングを行うことができる。 The area of the dyed portion is preferably the average value of the area calculated from a plurality of randomly selected ranges.
By using the average value of the areas of a plurality of locations as an index, more accurate screening can be performed.
　次いで、ステップＳ３０にて得られた面積を指標として、探索対象成分の接着タンパク質の増減への影響を評価する評価ステップＳ４０を行う。 Next, using the area obtained in step S30 as an index, evaluation step S40 is performed to evaluate the effect of the component to be searched for on the increase or decrease of the adhesive protein.
　図３に、ステップＳ４０を詳細に表したフローチャートを示す。図３を参照して、前記スクリーニング試料の染色部分の面積と、前記スクリーニング対照試料の染色部分の面積とを比較する一実施形態における、評価工程について説明する。 FIG. 3 shows a flowchart showing step S40 in detail. The evaluation step in one embodiment in which the area of the stained portion of the screening sample and the area of the stained portion of the screening control sample are compared with reference to FIG. 3 will be described.
　ステップＳ４１において、スクリーニング試料の染色部分の面積Ａｔと、スクリーニング試料の染色部分の面積Ａｃとを比較する。
　面積の比較は、面積の絶対値の比較であっても良く、スクリーニング対照試料の面積を１００％とした場合における面積の割合の比較であってもよい。
　複数のスクリーニング試料について評価を行う場合には、スクリーニング対照試料の面積を１００％とした比較を行うのが好ましい。 In step S41, the area At of the stained portion of the screening sample and the area Ac of the stained portion of the screening sample are compared.
The comparison of the areas may be a comparison of the absolute values of the areas, or may be a comparison of the ratio of the areas when the area of the screening control sample is 100%.
When evaluating a plurality of screening samples, it is preferable to make a comparison with the area of the screening control sample as 100%.
　面積Ａｔが面積Ａｃより小さい場合（面積Ａｔ　＜　面積Ａｃ）、ステップＳ４２に移行し、当該スクリーニング試料に添加した探索対象成分は、接着タンパク質の量を減少する作用を有する成分であると評価する。 When the area At is smaller than the area Ac (area At <area Ac), the process proceeds to step S42, and the search target component added to the screening sample is evaluated as a component having an action of reducing the amount of adhesive protein.
　一方で、面積Ａｔが面積Ａｃと同一、又は面積Ａｔが面積Ａｃより大きい高い場合（面積Ａｔ　≦　面積Ａｃ）にはステップＳ４３に移行し、当該スクリーニング試料に添加した探索対象成分は、接着タンパク質の量を減少する作用を有する成分であると評価する。 On the other hand, when the area At is the same as the area Ac or the area At is higher than the area Ac (area At ≤ area Ac), the process proceeds to step S43, and the search target component added to the screening sample is the adhesive protein. It is evaluated as a component having an action of reducing the amount.
　図３を参照して、一実施形態に係る評価工程について説明したが、本発明のスクリーニング方法における評価工程は、スクリーニング試料の染色部分の面積を指標とした、接着タンパク質の増減への影響の評価であれば、特に限定されない。 Although the evaluation step according to the embodiment has been described with reference to FIG. 3, the evaluation step in the screening method of the present invention uses the area of the stained portion of the screening sample as an index to evaluate the effect on the increase or decrease of the adhesive protein. If so, there is no particular limitation.
　例えば、接着タンパク質の量を増加する成分をスクリーニングしたい場合においては、ステップＳ４１にて、「面積Ａｔ　＞　面積Ａｃ」であるかを判定し、結果が「ｙｅｓ」であれば、ステップＳ４２にて、「接着タンパク質の量を増加する作用を有する成分である」と評価してもよい。
　また、ステップＳ４２において、「接着タンパク質の量を減少する作用を有する成分である」と評価し、ステップＳ４３において、「接着タンパク質の量を増加する作用を有する成分である」と評価してもよい。 For example, when it is desired to screen a component that increases the amount of adhesive protein, it is determined in step S41 whether "Area At> Area Ac", and if the result is "yes", in step S42. It may be evaluated as "a component having an action of increasing the amount of adhesive protein".
Further, in step S42, it may be evaluated as "a component having an action of reducing the amount of adhesive protein", and in step S43, it may be evaluated as "a component having an action of increasing the amount of adhesive protein". ..
　また、別の実施形態においては、ステップＳ２０で説明した通り、探索対象成分の濃度が異なる複数のスクリーニング試料を調製し、これらを評価する工程であってもよい。
　当該実施形態においては、複数のスクリーニング試料における面積を比較し、接着タンパク質の量が減少する作用、又は増加する作用において、顕著な効果が発現するしきい値となる特定の濃度が存する場合には、「当該探索対象成分は、特定の濃度以上で顕著な接着タンパク質の量が減少する作用、又は増加する作用を有する。」と評価することができる。
　また、特定の濃度以上としないと上記作用が発現しない成分については、「当該探索対象成分は、特定の濃度以上で接着タンパク質の量が減少する作用、又は増加する作用を発現する」と評価することができる。 Further, in another embodiment, as described in step S20, there may be a step of preparing a plurality of screening samples having different concentrations of the components to be searched and evaluating them.
In this embodiment, the areas of a plurality of screening samples are compared, and when there is a specific concentration that becomes a threshold value at which a remarkable effect is exhibited in the action of reducing or increasing the amount of adhesive protein. , "The component to be searched has an action of significantly reducing or increasing the amount of adhesive protein that is remarkable above a specific concentration."
In addition, for a component that does not exhibit the above action unless the concentration is above a specific concentration, it is evaluated that "the component to be searched exhibits an action of decreasing or increasing the amount of adhesive protein above a specific concentration". be able to.
　本発明のスクリーニング方法は、敏感肌の改善及び／又は予防のために用いられる成分のスクリーニングに用いることが好ましい。
　ここで、本明細書における「敏感肌」は、外部刺激（乾燥、紫外線、化合物の塗布）抵抗性が低下（皮膚刺激値が低下）した肌質をいう。 The screening method of the present invention is preferably used for screening components used for improving and / or preventing sensitive skin.
Here, the term "sensitive skin" as used herein refers to a skin type having reduced resistance to external stimuli (dryness, ultraviolet rays, application of compounds) (decreased skin stimulus value).
　より具体的には、本明細書における「敏感肌」は、（１）及び／又は（２）で提起される肌質をいう。 More specifically, "sensitive skin" in the present specification refers to the skin type raised in (1) and / or (2).
（１）「医薬品外用剤、化粧品、植物、紫外線、金属などの物質に反応し、皮膚トラブルを起こしやすい肌。また、アレルギー性物質（花粉、香料など）や刺激性物質（アルコールなど）に過敏な肌」
（２）「睡眠不足、過労、生理、季節の変わり目、精神的なストレスなどの状況下で、刺激物に対して一時的に皮膚トラブルを起こしやすくなる肌。」 (1) "Skin that reacts to substances such as external medicines, cosmetics, plants, ultraviolet rays, and metals and is prone to skin problems. Also, it is sensitive to allergic substances (pollen, fragrances, etc.) and irritating substances (alcohol, etc.). Skin "
(2) "Skin that is temporarily prone to skin problems due to irritants under conditions such as lack of sleep, overwork, menstruation, seasonal changes, and mental stress."
　また、本明細書における「敏感肌の改善及び／又は予防のため」は、外部刺激抵抗性が低下（皮膚刺激閾値が低下）した肌質の改善、及び／又は、肌質における外部刺激抵抗性の低下（皮膚刺激閾値の低下）の予防を指す。 In addition, "for improvement and / or prevention of sensitive skin" in the present specification means improvement of skin quality in which external stimulus resistance is reduced (skin stimulus threshold is lowered) and / or external stimulus resistance in skin quality. Refers to the prevention of a decrease in skin irritation (decrease in skin irritation threshold).
　また、本発明のスクリーニング方法は、ヒト表皮角化細胞の細胞間接着抑制作用を有する成分のスクリーニングに用いることが好ましい。
　なお、本明細書において、「細胞間接着」の語は、デスモゾーム蛋白質（デスモグレインを含むたんぱく質群）の増加による角層接着機能異常（細胞接着性の亢進により角層の重層化が進むこと）をいう（要すれば、特許４１９７１９４号、特許第４００２６３５号　参照）。
　また、本明細書において、「細胞間接着抑制」の概念は、細胞間接着の予防又は改善の何れも含む。 In addition, the screening method of the present invention is preferably used for screening a component having an intercellular adhesion inhibitory effect on human epidermal keratinocytes.
In addition, in this specification, the term "cell adhesion" refers to dysfunction of stratum corneum adhesion due to an increase in desmosome protein (protein group containing desmoglein) (progression of stratification of stratum corneum due to enhanced cell adhesion). (Refer to Japanese Patent No. 4197194 and Japanese Patent No. 4002635, if necessary).
Further, in the present specification, the concept of "inhibition of cell-cell adhesion" includes any of prevention or improvement of cell-cell adhesion.
　ここで、ヒト表皮角化細胞の細胞間接着が抑制されることで、角層の重層化が抑制され、角層重層剥離を抑制する作用を得ることができる。
　すなわち、本願発明のスクリーニング方法は、角層重層剥離の予防、又は改善作用を有する成分のスクリーニングに用いることができる。
　なお、本明細書において、「角層重層剥離」とは、角層が複数枚重なった状態で皮膚表面から剥がれ落ちることをいう。皮膚が角層重層剥離する状態か否かは、テープストリッピング法により角層を採取し、採取した角層を染色して観察することで確認することができる。 Here, by suppressing the cell-cell adhesion of human epidermal keratinocytes, the stratification of the stratum corneum is suppressed, and the effect of suppressing the exfoliation of the stratum corneum can be obtained.
That is, the screening method of the present invention can be used for screening components having a preventive or ameliorating effect on exfoliation of stratum corneum.
In the present specification, "exfoliation of stratum corneum" means exfoliation from the skin surface in a state where a plurality of stratum corneum are overlapped. Whether or not the skin is exfoliated from the stratum corneum can be confirmed by collecting the stratum corneum by the tape stripping method, staining the collected stratum corneum, and observing it.
　また、後述する実施例に示すように、肌の明度の低い箇所では、デスモグレイン１が多く存在し、肌の明度の低下は、角層の重層化によるものと考えられる。
　上記知見に鑑みると、デスモグレインを減少させる作用を有する成分は、肌の明度改善作用を有する成分であるということができる。
　すなわち、本発明のスクリーニング方法は、肌の明度改善作用を有する成分のスクリーニングに使用することができる。
　ここで、本明細書において、「肌の明度改善」は、正常状態の肌とデスモグレインの過剰存在により明度の低下した肌との間における、明度の差が少なくなることを指す。 Further, as shown in Examples described later, a large amount of desmoglein 1 is present in a place where the lightness of the skin is low, and it is considered that the decrease in the lightness of the skin is due to the stratification of the stratum corneum.
In view of the above findings, it can be said that the component having an action of reducing desmoglein is a component having an action of improving the lightness of the skin.
That is, the screening method of the present invention can be used for screening components having an effect of improving skin brightness.
Here, in the present specification, "improvement of lightness of skin" means that the difference in lightness between normal skin and skin whose lightness is reduced due to the excessive presence of desmoglein is reduced.
　また、後述する実施例に示すように、デスモグレイン減少作用を有する成分は、より透明感のある肌状態に改善する作用を有する。すなわち、本発明のスクリーニング方法は、肌の透明感向上作用を有する成分のスクリーニングに使用できる。 Further, as shown in Examples described later, a component having a desmoglein reducing action has an action of improving a more transparent skin condition. That is, the screening method of the present invention can be used for screening components having an effect of improving the transparency of the skin.
　また、後述する実施例に示すように、肌の硬度が高い箇所では、デスモグレイン１が多く存在し、肌の硬さの要因は、角層の重層化によるものと考えられる。
　上記知見に基づくと、デスモグレインを減少させることで、肌が柔らかくなる効果を得られる。すなわち、本発明のスクリーニング方法は、肌の軟化作用を有する成分のスクリーニングに使用できる。 Further, as shown in Examples described later, a large amount of desmoglein 1 is present in a place where the hardness of the skin is high, and it is considered that the factor of the hardness of the skin is due to the stratification of the stratum corneum.
Based on the above findings, reducing desmoglein can have the effect of softening the skin. That is, the screening method of the present invention can be used for screening components having a skin softening effect.
　ここで、肌が柔らかくなることにより、他の薬剤成分の浸透をより効率的に行うことができる。そのため、本発明のスクリーニング方法は、美白剤等の薬効成分をより効率良く肌に浸透させる作用を有する成分のスクリーニングに使用することができる。 Here, by softening the skin, it is possible to more efficiently permeate other drug components. Therefore, the screening method of the present invention can be used for screening a component having an action of more efficiently permeating a medicinal component such as a whitening agent into the skin.
［実施例１］
＜角層細胞試料の調製工程＞
　ヒト前腕外側より、セロハンテープで採取した角層を、スライドガラス（「ＭＡＳコート」、松浪硝子工業株式会社製）に転写し、当該スライドガラスをキシレンに一晩浸漬した。
　次いで、当該スライドガラスをキシレンで１０分間、２回洗浄した後、ＰＢＳで洗浄した。 [Example 1]
<Preparation process of stratum corneum cell sample>
From the outside of the human forearm, the stratum corneum collected with cellophane tape was transferred to a slide glass (“MAS coat”, manufactured by Matsunami Glass Industry Co., Ltd.), and the slide glass was immersed in xylene overnight.
The slide glass was then washed twice with xylene for 10 minutes and then with PBS.
＜探索対象成分の調製＞
　ワイルドタイム抽出液（一丸ファルコス社　製、ヨウシュイブキジャコウソウ（Ｗｉｌｄ　Ｔｈｙｍｅ，Ｔ．ｓｅｒｐｙｌｌｕｍ）の地上部抽出物）、及びトゲナシ抽出液（丸善製薬社　製、トゲナシ（ＲＯＳＡ　ｒｏｘｂｕｒｇｈｉｉ）の果実抽出物）を、それぞれ濃度が０．１質量％、０．０５質量％、０．０２５質量％、０．０１２５質量％となるようにＰＢＳで希釈し、探索対象成分を得た。
＜スクリーニング用試料の調製＞
　探索対象成分に、角層細胞を一晩浸漬し、試料を得た。また、対照試料として、探索対象成分に浸漬させない角層細胞を用意した。 <Preparation of search target components>
Wild thyme extract (above-ground extract of Wild Time, T. serpylum, manufactured by Ichimaru Falcos) and spiny extract (fruit extract of ROSA roxburghii, manufactured by Maruzen Pharmaceuticals Co., Ltd.) The components to be searched were obtained by diluting with PBS so that the concentrations were 0.1% by mass, 0.05% by mass, 0.025% by mass, and 0.0125% by mass, respectively.
<Preparation of screening sample>
A sample was obtained by immersing the stratum corneum cells in the component to be searched overnight. In addition, as a control sample, stratum corneum cells not immersed in the component to be searched were prepared.
＜免疫染色試験＞
　それぞれの試料をＰＢＳで洗浄後、４％ＰＦＡ・リン酸緩衝液中、室温で１５分間インキュベートした。
　次いで、それぞれの試料をＰＢＳで洗浄後、２０％ブロックエース（ＤＳバイオファーマメディカル社製）水溶液中、室温で１時間インキュベートした。
　それぞれの試料に、一次抗体（Ａｎｔｉ－Ｄｅｓｍｏｇｌｅｉｎ　１，Ｍｏｕｓｅ－Ｍｏｎｏ）を加え、湿潤箱中、室温で２時間インキュベートした。
　それぞれの試料をＰＢＳで５分間、３回洗浄した後、二次抗体（Ａｌｌｅｘａ　Ｆｌｕｏｒ＠４８８　Ｇｏａｔ　Ａｎｔｉ－ｍｏｕｃｅ　ＩｇＧ）、を加え、湿潤箱中、室温で１時間インキュベートした。
　それぞれの試料をＰＢＳで５分間、３回洗浄し、蒸留水で２回洗浄した後、Ｆｌｕｏｒｏｍｏｕｔ－Ｇ（ｓｏｕｔｈｅｒｎ　ｂｉｏｔｅｃｈ社製）を用いて封入し、複数のスクリーニング試料、及びスクリーニング対照試料を得た。 <Immunostaining test>
Each sample was washed with PBS and then incubated in 4% PFA phosphate buffer for 15 minutes at room temperature.
Then, each sample was washed with PBS and then incubated in a 20% aqueous solution of Block Ace (manufactured by DS Biopharma Medical) at room temperature for 1 hour.
A primary antibody (Anti-Dessmoglein 1, Mouse-Mono) was added to each sample, and the mixture was incubated in a wet box at room temperature for 2 hours.
After washing each sample with PBS for 5 minutes and 3 times, a secondary antibody (Allexa Fluor @ 488 Goat Anti-mouse IgG) was added, and the samples were incubated in a wet box at room temperature for 1 hour.
Each sample was washed with PBS for 5 minutes three times, washed twice with distilled water, and then encapsulated with Fluromout-G (manufactured by south biotech) to obtain a plurality of screening samples and a screening control sample. ..
＜画像解析＞
　スクリーニング試料、及びスクリーニング対照試料を、共焦点レーザー顕微鏡を用いて、それぞれ解析用画像を撮影した（図４）。
　画像解析ソフト（Ｉｍａｇｅ　Ｊ）を用いて、解析用画像における、４か所の任意の範囲を拡大、白黒画像化し、染色部分、及び非染色部分の面積を算出し、平均値を算出した。
　スクリーニング対照試料の染色部分の面積を１００％として、スクリーニング試料の染色部分の面積の割合（４か所の平均値）を算出した。結果を表１、図５に示す。 <Image analysis>
Images for analysis were taken of the screening sample and the screening control sample using a confocal laser scanning microscope (FIG. 4).
Using image analysis software (Image J), an arbitrary range of four places in the analysis image was enlarged and made into a black-and-white image, the areas of the stained part and the undyed part were calculated, and the average value was calculated.
The ratio of the area of the stained portion of the screening sample (the average value of the four locations) was calculated with the area of the stained portion of the screening control sample as 100%. The results are shown in Table 1 and FIG.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　図４及び図５に示す通り、ワイルドタイム抽出物、及びトゲナシ抽出物を添加したスクリーニング試料は、スクリーニング対照試料と比して、デスモグレイン１の発現量が減少していることがわかる。
　この結果から、ワイルドタイム抽出物、及びトゲナシ抽出物は、デスモグレイン１の量を減少する作用を有する成分であると評価することができる。 As shown in FIGS. 4 and 5, it can be seen that the expression level of desmoglein 1 was reduced in the screening sample to which the wild thyme extract and the spiny extract were added as compared with the screening control sample.
From this result, it can be evaluated that the wild thyme extract and the spiny extract are components having an action of reducing the amount of desmoglein 1.
　また、添加したワイルドタイム抽出物の濃度が０．１質量％であるスクリーニング試料は、濃度が０．０５、０．０２５、及び０．０１２５質量％であるスクリーニング試料と比して、染色部分の面積が約２．５倍～２．８倍減少していることがわかる。 In addition, the screening sample in which the concentration of the added wild thyme extract was 0.1% by mass was higher than that in the screening sample in which the concentrations were 0.05, 0.025, and 0.0125% by mass. It can be seen that the area is reduced by about 2.5 to 2.8 times.
　同様に、添加したトゲナシ抽出物の濃度が０．１質量％であるスクリーニング試料は、濃度が０．０５、０．０２５、及び０．０１２５質量％であるスクリーニング試料と比して、染色部分の面積が、７．６～８．０倍減少していることがわかる。 Similarly, the screening sample with a concentration of 0.1% by weight of the added spiny extract was compared to the screening sample with concentrations of 0.05, 0.025, and 0.0125% by weight of the stained portion. It can be seen that the area is reduced by 7.6 to 8.0 times.
　このことから、ワイルドタイム抽出物、及びトゲナシ抽出物は、その濃度が０．１質量％以上で、デスモグレイン１の量を減少する顕著な作用を有すると評価することができる。 From this, it can be evaluated that the wild thyme extract and the spiny extract have a remarkable effect of reducing the amount of desmoglein 1 at a concentration of 0.1% by mass or more.
［実施例２］
＜角層細胞試料の調製工程＞
　実施例１と同一の手法により、角層細胞試料を調製した。 [Example 2]
<Preparation process of stratum corneum cell sample>
A stratum corneum cell sample was prepared by the same method as in Example 1.
＜探索対象成分の調製＞
　ウスベニタチアオイ抽出液（ｊｕｒｌｉｑｕｅ社製、ウスベニタチアオイ（Ａｌｔｈａｅａ　Ｏｆｆｉｃｉｎａｌｉｓ）の根の抽出物）を、濃度が０．２５質量％となるようにＰＢＳで希釈し、探索対象成分を得た。 <Preparation of search target components>
The Marsh mallow extract (extract of the root of Marsh mallow (Althaea Officinalis) manufactured by jurlique) was diluted with PBS to a concentration of 0.25% by mass to obtain a component to be searched.
＜スクリーニング用試料の調製＞
　探索対象成分に、角層細胞を一晩浸漬し、試料を得た。また、対照試料として、探索対象成分に浸漬させない角層細胞を用意した。 <Preparation of screening sample>
A sample was obtained by immersing the stratum corneum cells in the component to be searched overnight. In addition, as a control sample, stratum corneum cells not immersed in the search target component were prepared.
＜免疫染色試験＞
　実施例１と同一の手法により、スクリーニング用試料、及びスクリーニング対照試料を得た。 <Immunostaining test>
A screening sample and a screening control sample were obtained by the same method as in Example 1.
＜画像解析＞
　実施例１と同一の手法により、スクリーニング用試料及びスクリーニング対照試料の解析用画像を撮影し、スクリーニング対照試料の染色部分の面積を１００％として、スクリーニング試料の染色部分の面積の割合）を算出した。結果を表２、図６、及び図７に示す。 <Image analysis>
An analysis image of the screening sample and the screening control sample was taken by the same method as in Example 1, and the area of the stained portion of the screening sample was set to 100%, and the ratio of the area of the stained portion of the screening sample) was calculated. .. The results are shown in Table 2, FIG. 6 and FIG.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　図６及び図７に示す通り、ウスベニタチアオイ抽出物を添加したスクリーニング試料は、スクリーニング対照試料と比して、デスモグレイン１の発現量が減少していることがわかる。
　この結果から、ウスベニタチアオイ抽出物は、デスモグレイン１の量を減少する作用を有する成分であると評価することができる。 As shown in FIGS. 6 and 7, it can be seen that the expression level of desmoglein 1 was reduced in the screening sample to which the marsh mallow extract was added as compared with the screening control sample.
From this result, it can be evaluated that the Marsh mallow extract is a component having an action of reducing the amount of desmoglein 1.
［実施例３］
　　以下、肌の明度の相違とデスモグレイン１の存在量の関係性及び、デスモグレイン１の存在量と肌の硬度の関係性を裏付ける試験結果を示す。 [Example 3]
The test results supporting the relationship between the difference in skin brightness and the abundance of desmoglein 1 and the relationship between the abundance of desmoglein 1 and the hardness of the skin are shown below.
（１）被験者及び測定部位
　本実施例では、４０歳から５２歳の女性２０名を被験者とした。
　そして、本実施例では、被験者顔面における、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）と正常状態の部位（図１３中部位Ｂ　参照）を測定部位として選定した。 (1) Subjects and measurement sites In this example, 20 women aged 40 to 52 years were used as subjects.
Then, in this example, a part of the subject's face where it was confirmed that the brightness was visually reduced (blackish part, see part A in FIG. 13) and a part in a normal state (see part B in FIG. 13). ) Was selected as the measurement site.
（２）測定
（２－１）L＊値、a＊値、b＊値測定
　上記の測定部位に分光光度計を当て、各部位５回ずつ測定に供することにより、L＊値、a＊値、b＊値を測定した。
　結果を、図８～図１０に示す。 (2) Measurement (2-1) L * value, a * value, b * value measurement By applying a spectrophotometer to the above measurement site and performing measurement 5 times for each site, L * value, a * value , B * values were measured.
The results are shown in FIGS. 8 to 10.
　L＊値に関し、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）は、正常状態の部位（図１３中部位Ｂ　参照）に比して、有意に低い値を示した。すなわち、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）は、正常状態の部位（図１３中部位Ｂ　参照）に比して、暗いことが確認できた。 Regarding the L * value, the part where it was confirmed that the brightness was visually reduced (blackish part, see part A in FIG. 13) was compared with the part in the normal state (see part B in FIG. 13). , Showed a significantly lower value. That is, the part where the brightness was visually confirmed to be reduced (blackish part, see part A in FIG. 13) was darker than the part in the normal state (see part B in FIG. 13). Was confirmed.
　a＊値に関し、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）は、正常状態の部位（図１３中部位Ｂ　参照）に比して、有意に高い値を示した。すなわち、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）は、正常状態の部位（図１３中部位Ｂ　参照）に比して、赤みが強いことが確認できた。 Regarding the a * value, the part where it was confirmed that the brightness was visually reduced (blackish part, see part A in FIG. 13) was compared with the part in the normal state (see part B in FIG. 13). , Showed a significantly higher value. That is, the part where the brightness was visually confirmed to be reduced (blackish part, see part A in FIG. 13) was reddish as compared with the part in the normal state (see part B in FIG. 13). I was able to confirm that it was strong.
　b＊値に関し、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）は、正常状態の部位（図１３中部位Ｂ　参照）に比して、有意に高い値を示した。すなわち、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）は、正常状態の部位（図１３中部位Ｂ　参照）に比して、黄みが強いことが確認できた。 b * Regarding the value, the part where it was confirmed that the brightness was visually reduced (blackish part, see part A in FIG. 13) was compared with the part in the normal state (see part B in FIG. 13). , Showed a significantly higher value. That is, the part where the brightness was visually confirmed to be reduced (blackish part, see part A in FIG. 13) was yellowish as compared with the part in the normal state (see part B in FIG. 13). Was confirmed to be strong.
（２－２）硬度測定
　上記（２－１）の試験に供した各測定部位にインデントメーターを押し当て５回ずつ測定に供することにより、硬度を測定した。
　結果を、図１１に示す。 (2-2) Hardness measurement The hardness was measured by pressing an indent meter against each measurement site subjected to the above test (2-1) and subjecting the measurement to 5 times each.
The results are shown in FIG.
　硬度に関し、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）は、正常状態の部位（図１３中部位Ｂ　参照）に比して、有意に低い値を示した。すなわち、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）は、正常状態の部位（図１３中部位Ｂ　参照）に比して、硬いことが確認できた。 Regarding the hardness, the part where it was confirmed that the brightness was visually reduced (blackish part, see part A in FIG. 13) was significant compared to the part in the normal state (see part B in FIG. 13). Showed a low value. That is, the part where it was confirmed that the brightness was visually reduced (blackish part, see part A in FIG. 13) was harder than the part in the normal state (see part B in FIG. 13). Was confirmed.
（２－３）デスモグレイン１（Ｄｓｇ１）染色
　上記（２－１）、（２－２）の試験に供した各測定部位の角層細胞をテープストリッピング法で採取し、スライドガラスに貼付した。 (2-3) Desmoglein 1 (Dsg1) Staining The stratum corneum cells of each measurement site used in the above tests (2-1) and (2-2) were collected by the tape stripping method and attached to a slide glass.
　角層細胞を貼付したスライドガラスを、キシレンに一晩浸漬させた。
　浸漬後、洗浄、風乾させたのち、４％パラホルムアルデヒド・リン酸緩衝液中で室温、条件下、１５分間静置した。 The slide glass to which the stratum corneum cells were attached was immersed in xylene overnight.
After immersion, it was washed and air-dried, and then allowed to stand in a 4% paraformaldehyde / phosphate buffer solution at room temperature for 15 minutes.
　１５分後、角層細胞をＰＢＳで洗浄し、０．５％ＴｒｉｔｏｎＸ（ナカライテスク社製）／ＰＢＳ溶液に、室温条件下、５分間浸漬させた。 After 15 minutes, the stratum corneum cells were washed with PBS and immersed in 0.5% Triton X (manufactured by Nacalai Tesque) / PBS solution under room temperature conditions for 5 minutes.
　浸漬後、洗浄し、ブロックエース水溶液中（ＤＳバイオファーマメディカル　ＵＫ－Ｂ８０）、室温条件下、１時間静置した。 After immersion, it was washed and allowed to stand in an aqueous block ace solution (DS Biopharma Medical UK-B80) under room temperature conditions for 1 hour.
　静置後、リキッドブロッカー（大道産業社　製）で枠付けし、一次抗体を加え、湿潤箱中、室温条件下、２時間静置した。
　一次抗体として、Ａｎｔｉ　Ｄｅｓｍｏｇｌｅｉｎ　１，Ｍｏｕｓｅ　Ｍｏｎｏ(Ｄｓｇ１　Ｐ２３)　Ｐｒｏｇｅｎ，６５１１１０　原液（ＩｇＧ　濃度：１０－１５ｇ／ｍＬ）を使用した。 After standing, the cells were framed with a liquid blocker (manufactured by Daido Sangyo Co., Ltd.), the primary antibody was added, and the mixture was allowed to stand in a wet box at room temperature for 2 hours.
Anti Desmoglein 1, Mouse Mono (Dsg1 P23) Progen, 651110 undiluted solution (IgG concentration: 10-15 g / mL) was used as the primary antibody.
　静置後、ＰＢＳを用い洗浄し、二次抗体を加え、湿潤箱中、室温条件下、１時間静置した。
　二次抗体として、Ａｌｌｅ　ｘａ　Ｆｌｕｏｒ＠４８８　Ｇｏａｔ　Ａｎｔｉ　ｍｏｕｓｅ　ＩｇＧ　Ｉｎｖｉｔｒｏｇｅｎ，Ａ　１１００１　ＰＢＳ２００倍希釈溶液を使用した。 After standing, the cells were washed with PBS, a secondary antibody was added, and the mixture was allowed to stand in a wet box under room temperature for 1 hour.
As a secondary antibody, Fluor xa Fluor @ 488 Goat Antimouse IgG Invitrogen, A 11001 PBS 200-fold diluted solution was used.
　静置後洗浄し、Ｆｌｕｏｒｏｍｏｕｔ－Ｇ（ｓｏｕｔｈｅｒｎ　ｂｉｏｔｅｃｈ　社製）で封入し、室温で３０分乾燥させた。 After standing, it was washed, sealed with Fluromout-G (manufactured by south biotech), and dried at room temperature for 30 minutes.
　乾燥後、マニキュアでカバーガラスを固定し、乾燥させたのち、（撮影）共焦点レーザー顕微鏡（Ｎｉｋｏｎ　Ａ１）、×２０での観察に供した。
　結果を、図１２に示す。 After drying, the cover glass was fixed with nail polish, dried, and then subjected to observation with a (photographed) confocal laser scanning microscope (Nikon A1) at × 20.
The results are shown in FIG.
　デスモグレイン１（Ｄｓｇ１）量に関し、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）は、正常状態の部位（図１３中部位Ｂ　参照）に比して、有意に高い値を示した。すなわち、目視により明度の低下していることが確認できた部位（黒みがかった部分、図１３中部位Ａ　参照）は、正常状態の部位（図１３中部位Ｂ　参照）に比して、デスモグレイン１（Ｄｓｇ１）量が多いことが確認できた。 Regarding the amount of desmoglein 1 (Dsg1), the part where it was visually confirmed that the brightness was reduced (blackish part, see part A in FIG. 13) is the part in the normal state (see part B in FIG. 13). The value was significantly higher than that of. That is, the part where the brightness was visually confirmed to be reduced (blackish part, see part A in FIG. 13) was desmoglein as compared with the part in the normal state (see part B in FIG. 13). It was confirmed that the amount of 1 (Dsg1) was large.
（３）考察
　図８～図１０、及び図１２に示すように、肌の明度の低い箇所では、デスモグレイン１が多く存在することが分かった。ここで、肌の明度の低下は、肌のｐＨが低くデスモグレイン１を分解するセリンプロテアーゼの活性が低いために、角層の重なりが密になったこと（角層の重層化）によるものと考えられる。 (3) Discussion As shown in FIGS. 8 to 10 and 12, it was found that a large amount of desmoglein 1 was present in places where the lightness of the skin was low. Here, the decrease in skin lightness is due to the fact that the pH of the skin is low and the activity of serine protease that decomposes desmoglein 1 is low, so that the stratum corneum is densely overlapped (layering of the stratum corneum). Conceivable.
　そして、前述の通り、本願発明の有効成分はデスモグレイン減少作用を奏する。
　上記知見に鑑みると、本発明の有効成分は、デスモグレイン１を減少させることによる肌の明度改善作用を奏することがわかった。 Then, as described above, the active ingredient of the present invention exerts a desmoglein reducing action.
In view of the above findings, it was found that the active ingredient of the present invention exerts an effect of improving the brightness of the skin by reducing desmoglein 1.
　図１１～図１２に示すように、肌の硬度が高い箇所では、デスモグレイン１が多く存在するが分かった。ここで、肌の硬さの要因は、肌のｐＨが低くデスモグレイン１を分解するセリンプロテアーゼの活性が低いために、角層の重なりが密になったこと（角層の重層化）によるものと考えられる。 As shown in FIGS. 11 to 12, it was found that a large amount of desmoglein 1 was present in places where the skin hardness was high. Here, the cause of the hardness of the skin is that the pH of the skin is low and the activity of the serine protease that decomposes desmoglein 1 is low, so that the stratum corneum is densely overlapped (layering of the stratum corneum). it is conceivable that.
　上記知見に基づくと、デスモグレイン１を減少させることにより、肌が柔らかくなることがわかった。
　そして、前述の通り、本願発明の有効成分はデスモグレイン減少作用を奏する。
　つまり、本発明の有効成分は、デスモグレイン１を減少させることによる肌の軟化作用を奏することがわかった。 Based on the above findings, it was found that reducing desmoglein 1 softens the skin.
Then, as described above, the active ingredient of the present invention exerts a desmoglein reducing action.
That is, it was found that the active ingredient of the present invention exerts a skin softening effect by reducing desmoglein 1.
［実施例４］
　以下、デスモグレイン減少作用を有する成分を含む組成物の重層剥離抑制作用の検証結果を示す。
（１）製剤の調製
　表３に記載の組成に従い、トゲナシ抽出物を含む製造例１の化粧料を調製した。 [Example 4]
Hereinafter, the verification results of the layer peeling inhibitory effect of the composition containing the component having a desmoglein reducing effect will be shown.
(1) Preparation of preparation According to the composition shown in Table 3, the cosmetic of Production Example 1 containing the spiny extract was prepared.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（２）被験者及び対象部位
　本実施例では、女性５名を被験者とし、被験者の左右前腕内側部（ｎ＝１０）に、製造例１の化粧品を１日２回（朝と晩）塗布し、これを１０日間続けた。 (2) Subjects and Target Sites In this example, five female subjects were used, and the cosmetics of Production Example 1 were applied to the medial parts of the left and right forearms (n = 10) of the subjects twice a day (morning and evening). This was continued for 10 days.
（３）デスモグレイン減少作用の検証
　製造例１の化粧品の使用前、使用後（１０日間継続後）のそれぞれの段階で、実施例１の「＜角層細胞試料の調製工程＞」に記載の方法に準拠して、各被験死者の左右前腕内側部の角層を採取し、角層細胞試料を調製した。次いで、実施例１の「＜免疫染色試験＞」に記載の方法に準拠し、角層細胞試料中のデスモグレイン１を染色し、共焦点レーザー顕微鏡を用いて、製造例１の使用前後の解析用画像を撮影し、蛍光（染色）の確認を行った。 (3) Verification of Desmoglein Reducing Effect Described in "<Step of preparing stratum corneum cell sample>" of Example 1 at each stage before and after using the cosmetic product of Production Example 1 (after continuing for 10 days). According to the method, the stratum corneum on the medial side of the left and right forearms of each test dead was collected to prepare a stratum corneum cell sample. Next, according to the method described in "<Immunostaining test>" of Example 1, desmoglein 1 in a stratum corneum cell sample was stained, and analysis before and after use of Production Example 1 was performed using a confocal laser scanning microscope. An image was taken and the fluorescence (staining) was confirmed.
　解析用画像全体の面積を１００％として、蛍光部分の面積を算出した。結果を図１４、図１５に示す。なお、図１５において、＊は、ｐ＜０．０５である。 The area of the fluorescent part was calculated with the area of the entire analysis image as 100%. The results are shown in FIGS. 14 and 15. In FIG. 15, * is p <0.05.
　図１４、及び図１５に示す通り、トゲナシ抽出物を含む製造例１の化粧品を使用し続けることで、デスモグレイン１の量が減少することが確認できた。 As shown in FIGS. 14 and 15, it was confirmed that the amount of desmoglein 1 was reduced by continuing to use the cosmetic product of Production Example 1 containing the spiny extract.
（４）重層剥離抑制作用の検証
　製造例１の化粧品の使用前後において、実施例１の「＜角層細胞試料の調製工程＞」に記載の方法に準拠して、各被験死者の左右前腕内側部の角層を採取し、角層細胞試料を調製した。次いで、それぞれの角層細胞試料を、ゲンチアナバイオレットとブリリアントグリーンを用いて常法により染色し、光学顕微鏡により観察した。
　結果の代表図を図１６に示す。 (4) Verification of multi-layer peeling inhibitory effect Before and after the use of the cosmetic product of Production Example 1, the inside of the left and right forearms of each test dead according to the method described in "<Step of preparing a stratum corneum cell sample>" of Example 1. The stratum corneum was collected and a stratum corneum cell sample was prepared. Next, each stratum corneum cell sample was stained by a conventional method using Gentian violet and brilliant green, and observed with an optical microscope.
A representative diagram of the results is shown in FIG.
　図１６に示す通り、製造例１の使用前における角層細胞の光学顕微鏡像には、色が濃い部分が複数存在している。この色濃い部分は、テープストリッピング法により、角層細胞が重層剥離した部分である。
　一方で、製造例１を１０日間使用し続けた後に採取した角層細胞試料では、使用前に採取した角層細胞試料と比して、大部分が淡く染色している。この淡い部分は、角層細胞が一層のみ剥がされた部分であり、すなわち、重層剥離が生じていないことを意味している。
　以上の通り、デスモグレイン減少作用を有する成分を含む製造例１の化粧品を使用することで、重層剥離が改善されることが確認できた。 As shown in FIG. 16, the optical microscope image of the stratum corneum cells before use of Production Example 1 has a plurality of dark-colored portions. This dark part is the part where the stratum corneum cells have been exfoliated by the tape stripping method.
On the other hand, most of the stratum corneum cell samples collected after using Production Example 1 for 10 days are lightly stained as compared with the stratum corneum cell samples collected before use. This pale part means that only one layer of stratum corneum cells has been exfoliated, that is, no multi-layer exfoliation has occurred.
As described above, it was confirmed that the multi-layer peeling was improved by using the cosmetic product of Production Example 1 containing a component having a desmoglein reducing effect.
［実施例５］
　以下、デスモグレイン減少作用を有する成分を含む組成物のデスモグレイン減少能を調べた。 [Example 5]
Hereinafter, the desmoglein-reducing ability of a composition containing a component having a desmoglein-reducing effect was investigated.
（１）組成物の調製
　下記表４に示す処方に従って、ワイルドタイム抽出物を含む化粧料（乳液）を調製した。即ち、（イ）、（ロ）、（ハ）の成分をそれぞれ７０℃に加熱し、（ロ）に（ハ）を加えて中和し、これを撹拌しながら（イ）を徐々に加えて乳化し、ホモジナイザーを用いて均質化した後、撹拌しながら冷却し、製造例２の乳液を得た。 (1) Preparation of composition A cosmetic (milky lotion) containing a wild thyme extract was prepared according to the formulation shown in Table 4 below. That is, each of the components (a), (b), and (c) is heated to 70 ° C., (c) is added to (b) to neutralize, and (a) is gradually added while stirring this. After emulsification and homogenization using a homogenizer, the mixture was cooled with stirring to obtain the emulsion of Production Example 2.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
（２）被験者及び測定部位
　本実施例では、４０歳から５２歳の女性２０名を被験者とした。
　そして、本実施例では、被験者顔面における、シミが存在する部位（９２箇所）を測定部位として選定した。
　各被験者のシミが存在する部位に、製造例１の乳液を１日２回塗布し、これを１２週間続けた。 (2) Subjects and measurement sites In this example, 20 women aged 40 to 52 years were used as subjects.
Then, in this example, the sites (92 sites) where the stains are present on the subject's face were selected as the measurement sites.
The emulsion of Production Example 1 was applied twice a day to the spots of each subject, and this was continued for 12 weeks.
（３）測定
　製造例の乳液の使用前、使用継続６週間経過時点、及び１２週間経過時点において、実施例１の「＜角層細胞試料の調製工程＞」に記載の方法に準拠して、被験者のシミが存在する部位の角層細胞を採取し、角層細胞試料を調製した。次いで、実施例１の「＜免疫染色試験＞」に記載の方法により準拠して、それぞれの角層細胞試料におけるデスモグレイン１を染色し、共焦点レーザー顕微鏡を用いて、製造例２の使用前後の解析用画像を撮影し、蛍光（染色）の確認行った。 (3) Measurement Before using the emulsion of the production example, after 6 weeks of continuous use, and after 12 weeks of use, in accordance with the method described in "<Step of preparing stratum corneum cell sample>" of Example 1, according to the method described in Example 1. The stratum corneum cells at the site where the subject's stains were present were collected, and a stratum corneum cell sample was prepared. Then, according to the method described in "<Immunostaining test>" of Example 1, desmoglein 1 in each stratum corneum cell sample was stained, and before and after the use of Production Example 2 using a confocal laser scanning microscope. An image for analysis was taken and the fluorescence (staining) was confirmed.
　解析用画像全体の面積を１００％として、蛍光部分の面積を選出した。結果を表５、表６、及び図１７（代表）、図１８、１９に示す。なお、乳液使用１２週間経過後のデータは、シミ部位９３箇所のうち、８０箇所のデータを集計し、同箇所における乳液使用前のデータと比較した。また、図１８において、＊＊はｐ＜０．０１であり、図１９において、＊＊＊はｐ＜０．００１である。 The area of the fluorescent part was selected with the area of the entire analysis image as 100%. The results are shown in Tables 5, 6, and 17 (representative), 18 and 19. As for the data after 12 weeks of using the emulsion, the data of 80 of the 93 spots were aggregated and compared with the data before using the emulsion at the same site. Further, in FIG. 18, ** is p <0.01, and in FIG. 19, *** is p <0.001.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　表５～６、及び図１７～１９に示す通り、ワイルドタイム抽出物を含む乳液を継続的に使用することで、シミ部位におけるデスモグレイン１量が有意に減少することが確認できた。 As shown in Tables 5 to 6 and FIGS. 17 to 19, it was confirmed that the amount of desmoglein 1 at the spot site was significantly reduced by continuous use of the emulsion containing the wild thyme extract.
　本発明は、接着タンパク質の量に関連する解析手法に応用できる。

  The present invention can be applied to analytical methods related to the amount of adhesive protein.

Claims (8)

  1. 　角層細胞間の接着タンパク質の増減に影響を与える成分のスクリーニング方法であって、
    　テープストリッピング法を用いて、角層細胞を採取する採取工程と、
    　表面が正電荷に帯電したスライドガラス上に、角層を転写する転写工程と、
    　前記スライドガラスを洗浄する洗浄工程と、
    　前記スライドガラスに探索対象成分を添加し、免疫組織染色法により、接着タンパク質を染色し、スクリーニング試料を調製する調製工程と、
    　前記スクリーニング試料の任意の箇所を撮影し、解析用画像を得る画像取得工程と、
    　前記解析用画像における染色部分の面積を指標として、前記探索対象成分の接着タンパク質の増減への影響を評価する評価工程を備える、スクリーニング方法。     A screening method for components that affect the increase or decrease of adhesive proteins between stratum corneum cells.
    A collection process for collecting stratum corneum cells using the tape stripping method,
    A transfer process that transfers the stratum corneum onto a glass slide whose surface is positively charged,
    A cleaning process for cleaning the slide glass and
    A preparation step of adding a component to be searched for to the slide glass, staining an adhesive protein by an immunohistochemical staining method, and preparing a screening sample.
    An image acquisition step of photographing an arbitrary part of the screening sample to obtain an image for analysis, and
    A screening method comprising an evaluation step of evaluating the influence of the component to be searched on the increase / decrease of the adhesive protein using the area of the stained portion in the analysis image as an index.
  2. 　前記調製工程において、前記探索対象成分を添加せずにスクリーニング対照試料を調製し、
    　前記評価工程が、前記スクリーニング試料の染色部分の面積と、前記スクリーニング対照試料の染色部分の面積とを比較して、前記スクリーニング試料の染色部分の面積が小さい場合、前記探索対象成分は接着タンパク質の量を減少する作用を有すると評価する工程である、請求項１に記載のスクリーニング方法。     In the preparation step, a screening control sample was prepared without adding the component to be searched.
    When the evaluation step compares the area of the stained portion of the screening sample with the area of the stained portion of the screening control sample and the area of the stained portion of the screening sample is small, the search target component is the adhesive protein. The screening method according to claim 1, which is a step of evaluating having an action of reducing the amount.
  3. 　前記調製工程において、前記探索対象成分の種類が同一であって、濃度が異なる複数のスクリーニング試料を調製し、
    　前記評価工程が、複数の前記スクリーニング試料の染色部分の面積を比較して、接着タンパク質の量を減少する作用における最適濃度を評価する工程である、請求項１に記載のスクリーニング方法。     In the preparation step, a plurality of screening samples having the same type of the component to be searched but having different concentrations are prepared.
    The screening method according to claim 1, wherein the evaluation step is a step of comparing the areas of the stained portions of the plurality of screening samples to evaluate the optimum concentration in the action of reducing the amount of adhesive protein.
  4. 　前記接着タンパク質が、デスモグレイン１（ｄｅｓｍｏｇｌｅｉｎ１）である、請求項１～３の何れか一項に記載のスクリーニング方法。 The screening method according to any one of claims 1 to 3, wherein the adhesive protein is desmoglein1.
  5. 　前記スライドガラスが、表面にＮＨ３ ＋が配列されたスライドガラスである、請求項１～４の何れか一項に記載のスクリーニング方法。 Said slide glass, NH 3 + is a glass slide which is arranged on the surface, the screening method according to any one of claims 1 to 4.
  6. 　前記洗浄工程が、前記スライドガラスをキシレンに浸漬する工程である、請求項１～５の何れか一項に記載のスクリーニング方法。 The screening method according to any one of claims 1 to 5, wherein the cleaning step is a step of immersing the slide glass in xylene.
  7. 　テープストリッピング法を用いた角層細胞試料の調製方法であって、
    　表面が正電荷に帯電したスライドガラス上に、角層を転写する転写工程と、
    　前記スライドガラスを洗浄する洗浄工程と、を備える、角層細胞試料の調製方法。     A method for preparing a stratum corneum cell sample using the tape stripping method.
    A transfer process that transfers the stratum corneum onto a glass slide whose surface is positively charged,
    A method for preparing a stratum corneum cell sample, comprising a washing step of washing the slide glass.
  8. 　前記スライドガラスが、表面にＮＨ３ ＋が配列されたスライドガラスである、請求項７に記載の調製方法。

          The slide glass is a glass slide NH 3 + are arranged in a surface preparation method according to claim 7.
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